Considerable progress has been made in defining the transcriptional events that control the stepwise Differentiation of unspecified neural precursors into motor neurons that innervate specific muscle targets. Despite these advances, much remains to be learned of the transcriptional regulatory network that subtends this process. Based on the ability to generate homogeneous preparations of specific motor neuron subtypes from ES cells, this project will take a global approach to defining transcriptional differences between motor neurons and other spinal neurons. Transcription factors identified will then be used to identify target genes and thereby iteratively define transcriptional networks. The work is structured around three aims. Aim 1. will use transcription factors known to determine motor neuron or dorsal interneuron fate to drive ES cell differentiation, in order to identify factors potentially involved in the acquisition of generic motor neuron identity. Aim 2. will use intrinsic and extrinsic factors that drive the differentiation of motor neurons characteristic of specific columns or pools from mouse ES cells, to define the transcrptional logic that controls motor neuron subtype identity. These basic advances will be applied in Aim 3. to the study of Spinal Muscular Atrophy (SMA), a developmental disease that results from reduction in levels of a protein, SMN, that is required for motor neuron survival. New ES cell lines will be derived from motor neurons expressing normal and reduced levels of SMN, obtained from mouse models for SMA. These disease-specific ES cells will be screened for differences in transcriptional repertoire. Functional testing of genes identified as potential effectors in these screens will be performed using a panel of in vitro and in vivo test systems focused on motor neuron differentiation, survival and axon growth. The project should also provide new approaches to potential therapeutic targets in SMA. Relevance Studying the way in which specific groups of motor neurons in the spinal cord develop to innervate specific muscles is central to understanding how precise control of breathing and movement is achieved. These studies will provide clues for understanding why specific groups of motor neurons degenerate and die in patients with diseases such as spinal muscular atrophy (SMA). This project will identify molecular mechanisms involved both in normal development and pathologic degeneration of this important neuronal population.